now detected throughout pachynema (Fig. 4, A
and B, and fig. S10). Analysis of the Rnf212–/–
Hei10–/– double mutant revealed that this hyper-SUMO phenotype was RNF212 dependent (Fig.
4, A and B). Thus, RNF212 and HEI10 mediate,
respectively, formation and turnover of axis-associated SUMO conjugates. Although the fungus,
Sordaria macrospora, lacks a RNF212 homolog,
its HEI10-like protein has also been shown to
modulate SUMO levels along SCs (23).

Consistent with a model in which RNF212-
mediated SUMO conjugation creates substrates
for HEI10-dependent proteasomal degradation,
axis-localized ubiquitin and the recruitment of
proteasomes were diminished in Rnf212–/– and
Hei10–/– single mutants and the Rnf212–/– Hei10–/–
double mutant (Fig. 4, C to F, and fig. S10);
ubiquitin staining of sex chromosomes appeared
unaffected. Analogous observations were made
for female meiosis (fig. S11). Together, these results point to the same pathway relationships
inferred from inhibitor studies, with RNF212-
dependent SUMOylation establishing a requirement for ubiquitin-dependent proteolysis via HEI10.
However, unlike general inhibition of the SMS
and UPS, Rnf212–/– and Hei10–/– mutations do
not cause overt defects in synapsis or progression
to metaphase (21, 22). Thus, the SUMO-ubiquitin-proteasome relay defined by RNF212 and HEI10
appears dedicated to the regulation of post-synapsis steps of meiotic recombination.

To better understand how recombination is
regulated by the RNF212-HEI10 pathway, we
analyzed the same set of markers described
above for chemical inhibition experiments (Fig.
4, G to L, and figs. S12 and S13). Although pan-inhibition caused abnormally high numbers of
MEI4 foci to persist through zygotene (Fig. 3, A
and B), mutation of Rnf212 and/or Hei10 did not
(Fig. 4, G and H) indicating that MEI4 turnover
does not require the RNF212-HEI10 pathway. In
contrast, RAD51 turnover was strongly dependent on HEI10; in Hei10–/– nuclei, high numbers
of RAD51 foci persisted throughout zygonema
and pachynema (Fig. 4, I and J, and fig. S12).
Moreover, persistence of RAD51 was RNF212
dependent as foci decreased to wild-type levels
in the Rnf212–/– Hei10–/– double mutant. Unlike
global UPS inhibition, which impeded turnover
of both RAD51 and DMC1, Hei10–/– mutation
did not slow the disappearance of DMC1 foci
(fig. S12). Thus, turnover of the two RecA homologs involves distinct branches of the UPS,
distinguished by their dependency on HEI10.
This result is consonant with studies showing
that DMC1 is the dominant DNA strand-exchange
activity during meiosis, whereas RAD51 plays an
essential supporting role that does not require its
catalytic activity (24, 25). Differential regulation
of DMC1 and RAD51 by the RNF212-HEI10 pathway suggests that RAD51 also plays a later role in
meiotic recombination.

We extended our previous studies showing
that Rnf212 and Hei10 mutations differentially
affect turnover of the ZMM factor, MutSg, with
faster turnover in the absence of RNF212 and
persistence when HEI10 was absent (21, 22)

(fig. S13). Differential effects of Rnf212–/– and

Hei10–/– mutations were also observed for MER3

(Fig. 4, K and L) and a third ZMM factor, TEX11
(fig. S13). In all three cases, persistence of high
numbers of ZMM foci seen in Hei10–/– mutant
nuclei was largely RNF212 dependent. We also
confirmed that the general DSB marker, gH2AX,
persists along synapsed chromosomes of Hei10–/–
spermatocytes (21) and showed that persistence
was dependent on RNF212 (fig. S13).

Although crossing over is abolished in Rnf212–/–
and Hei10–/– mutants, overall DSB repair remains
efficient (21, 22). Moreover, unlike pan-inhibition
of SUMO and ubiquitin conjugation (Fig. 2),
Rnf212–/– and Hei10–/– mutations do not cause
synapsis defects, which could cause secondary
defects in recombination. These considerations
support direct roles for the SMS and UPS in
regulating recombination. Consistently, subsets
of RNF212 and HEI10 foci precisely localize to
recombination sites (21, 22). However, costaining for the general recombination marker, RPA,
and SUMO, ubiquitin, or proteasomes revealed
only 28 to 38% colocalization (fig. S14). Thus,
the bulk of chromosomal SUMO, ubiquitin,
and proteasomes does not stably localize with
recombination sites, suggesting that the branch
of the SUMO-UPS pathway defined here may
regulate recombination indirectly via chromosome axes. That said, most RPA foci localized
immediately adjacent to, or interdigitated with,
SUMO, ubiquitin, or proteasome signals (fig. S14).
Moreover, modification of recombination factors by SUMO or ubiquitin and interaction with
proteasomes are expected to be transient.

In conclusion, the SMS and UPS function
coordinately to facilitate major transitions of
meiotic prophase including axis morphogenesis,
homolog synapsis and recombination (fig. S15).
RNF212 and HEI10 define an axis-associated,
SUMO-ubiquitin-proteasome relay that mediates
turnover of the subset of recombination factors
that act after DMC1-promoted homolog pairing.
This stage marks a key regulatory transition
during which a small number of crossover sites
are designated from the large pool of ongoing
recombination events in such a way that each
chromosome pair acquires at least one chiasma
(fig. S15) (26). We suggest that RNF212-mediated
SUMOylation establishes a precondition for this
crossover or noncrossover differentiation process by rendering the turnover of key recombination factors contingent on HEI10-dependent
proteolysis. At most recombination sites, HEI10-
targeted proteolysis predominates to destabilize
nascent intermediates and promote a noncrossover outcome. Designation of a crossover
outcome ensues at a minority of sites where
RNF212-dependent stabilization predominates,
enabling crossover-specific events, including formation of double-Holliday junctions and recruitment of factors such as MutLg (22). Absent the
RNF212-HEI10 pathway, recombination factors
that would normally experience transitory stabilization instead turn over more rapidly, crossover designation does not occur, and all DSBs
are repaired with a noncrossover outcome.

Our model is consonant with the possibility
that HEI10 is a SUMO-targeted ubiquitin ligase
(STUbL). Indeed, the RNF212-HEI10 pathway
shares general features with STUbL pathways
that regulate DSB repair in somatic cells (27).
These pathways stall DSB repair at an early step
until appropriate features have been installed
that minimize aberrant repair (28). We suggest
that SUMO conjugation may be a common
checkpoint-like mechanism to stall biological
processes to enable implementation of important regulatory decisions.

We thank A. Gomes, B. de Massy, C. Höög, and V. Dixit for
antibodies; M. Paddy for assistance with SIM; and the Hunter Lab
for support and discussions. This work was supported in part by
National Institute of General Medical Sciences, NIH, grant GM084955.